Each cell cycle, a human cell must specifically initiate DNA replication at multiple sites and accurately duplicate the entire 4x10-9 base pair human genome. Perturbations of chromosomal DNA replication lead to abnormal growth and are associated with various disease states. The goal of this research is to elucidate molecular mechanisms and regulatory processes of chromosomal DNA replication. Because the process of chromosomal replication is complex, we will use the virus, simian virus 40 (SV40) as a model system for examining the mechanism of eukaryotic DNA replication. With the exception of the virally encoded initiator large T antigen, SV40 relies completely on the host replication apparatus, so it provides an excellent system for the biochemical analysis of cellular replication proteins and processes. This proposal focuses on the human multi-subunit, single-stranded DNA- binding protein, Replication Protein A (hRPA). This protein is essential for DNA replication. In addition, hRPA is involved in DNA repair and recombination, and may be a target for regulation in these processes. hRPA binds tightly to single-strand DNA, affects the activity of other replication proteins and interacts specifically with large T antigen, DNA polymerase alpha, the tumor suppressor p53, several transcriptional initiators (e.g. GAL4 and VP16). The physiological importance of the activities attributed to hRPA and the function of hRPA in the cell are currently not understood. The objective of this research is to determine the specific role of hRPA in DNA replication. A combination of biochemical assays and mutational analysis will be used to elucidate the function of hRPA. The specific functions of each of the three subunits of hRPA and the structure of hRPA will be determined. The interactions of hRPA with both DNA and other proteins will be studied and quantitated to determine their importance for hRPA function. hRPA becomes phosphorylated at the beginning of S-phase. The role of post-translational modifications on hRPA function in DNA replication will be determined.